OBJECTIVE: To investigate the protective effect of dexmedetomidine (DEX) against erastin-induced ferroptosis in human renal tubular epithelial cells (HK-2 cells) and explore the underlying mechanism.
METHODS: HK-2 cells were treated with erastin alone or in combination with different concentrations (2.5, 5.0 and 10 μmol/L) of DEX, and the changes in cell viability were observed using CCK-8 assay. To explore the mechanism by which DEX inhibits erastin-induced ferroptosis, HK-2 cells were treated with erastin, erastin+10 μmol/L DEX, or erastin+10 μmol/L DEX+ML385 (a Nrf2 inhibitor), after which the cell viability was assessed. The level of intracellular Fe2+ was detected by cell ferrous iron colorimetric assay kit, and flow cytometry was performed to detect reactive oxygen species (ROS); MDA and reduced glutathione assay kits were used to detect the contents of MDA and GSH in the cells; The expressions of Nrf2, HO-1 and GPX4 proteins were detected by Western blotting.
RESULTS: Erastin treatment significantly inhibited the viability of the cells, decreased GSH content, and increased intracellular levels of Fe2+, ROS and MDA. The combined treatment with 10 μmol/L DEX markedly increased the viability of the cells, increased GSH content, reduced the levels of Fe2+, ROS and MDA, and upregulated the protein expressions of Nrf2, HO-1 and GPX4 in the cells. The application of ML385 obviously blocked the protective effect of DEX and caused significant inhibition of the Nrf2/HO-1/GPX4 pathway, decreased the cell viability and GSH content, and increased the levels of Fe2+, ROS and MDA in HK-2 cells.
CONCLUSIONS: The protective effect of DEX against erastin-induced ferroptosis of HK-2 cells is probably mediated by activation of the Nrf2/HO-1/GPX4 pathway to inhibit oxidative stress.

Background and Objectives: Selenium deficiency represents a risk factor for the occurrence of severe diseases, such as acute kidney injury (AKI). Recently, selenoprotein-p1 (SEPP1), a selenium transporter, mainly released by the liver, has emerged as a promising plasmatic biomarker of AKI as a consequence of cardio-surgery operations. The aim of the present study was to investigate, on an in vitro model of hypoxia induced in renal tubular cells, HK-2, the effects of sodium selenite (Na2SeO3) and to evaluate the expression of SEPP1 as a marker of injury. Materials and Methods: HK-2 cells were pre-incubated with 100 nM Na2SeO3 for 24 h, and then, treated for 24 h with CoCl2 (500 µM), a chemical hypoxia inducer. The results were derived from an ROS assay, MTT, and Western blot analysis. Results: The pre-treatment determined an increase in cells\' viability and a reduction in reactive oxygen species (ROS), as shown by MTT and the ROS assay. Moreover, by Western blot an increase in SEPP1 expression was observed after hypoxic injury as after adding sodium selenite. Conclusions: Our preliminary results shed light on the possible role of selenium supplementation as a means to prevent oxidative damage and to increase SEPP1 after acute kidney injury. In our in vitro model, SEPP1 emerges as a promising biomarker of kidney injury, although further studies in vivo are necessary to validate our findings.

OBJECTIVE: To investigate the role of SPP1 gene in acute kidney injury induced by renal ischemia-reperfusion injury (IRI).
METHODS: Twelve Sprague-Dawley rats were randomly divided into sham group and IRI group (n=6) and subjected to sham operation and renal ischemia for 30 min induced by penal pedicle clamping using non-traumatic microvascular clamps, respectively.Serum creatinine and blood urea nitrogen levels were detected, and PAS staining was used for pathological examination of the kidneys in the two groups.The renal expressions of SPP1, α-SMA and caspase-3 were detected using immunohistochemistry and immunofluorescent staining.In cultured renal tubular epithelial cells (HK-2 cells), Western blotting was performed to detect the changes in expressions of SPP1, caspase-3, and Kim-1 proteins following hypoxiareoxygenation (H/R) and transfection with si-NC or si-SPP1;flow cytometry was employed to analyze apoptosis of the treated cells.
RESULTS: Renal IRI caused significant elevations of serum creatinine and blood urea nitrogen levels (P<0.05) and induced severe shedding and necrosis of the renal tubular epithelial cells in the rats, resulting also in significantly up-regulated renal expressions of SPP1, α-SMA and caspase-3(P<0.05).In HK-2 cells, H/R significantly increased the protein expression levels of SPP1, caspase-3, and Kim-1(P<0.05), and compared si-NC transfection, transfection with SPP1 obviously reduced caspase-3 and Kim-1 expressions and lowered apoptosis rate of the cells with H/R exposure (P<0.05).
CONCLUSIONS: SPP1 is up-regulated in the kidneys of rats with renal IRI, and down-regulation of SPP1 expression can inhibit H/R-induced apoptosis of renal tubular epithelial cells.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

UNASSIGNED: We established a diquat-induced human kidney-2 cells (HK-2 cells) apoptosis model in this study to identify differentially expressed microRNAs (miRNAs) and signaling pathways involved in diquat poisoning via gene sequencing and bioinformatics analysis and explored the related therapeutic benefits.
UNASSIGNED: The effects of diquat on the viability and apoptosis of HK-2 cells were explored using the CCK-8 and Annexin V-FITC/PI double staining methods. Total RNAs were extracted using the TRizol method and detected by Illumina HiSeq 2500. Bioinformatics analysis was performed to explore differentially expressed (DE) miRNAs, their enriched biological processes, pathways, and potential target genes. The RT-qPCR method was used to verify the reliability of the results.
UNASSIGNED: Diquat led to HK-2 cell injury and apoptosis played an important role, hence an HK-2 cell apoptosis model in diquat poisoning was established. Thirty-six DE miRNAs were screened in diquat-treated HK-2 cells. The enriched biological process terms were mainly cell growth, regulation of apoptotic signaling pathway, extrinsic apoptotic signaling pathway, and Ras protein signal transduction. The enriched cellular components were mainly cell-cell junction, cell-substrate junction, ubiquitin ligase complex, and protein kinase complex. The enriched molecular functions were mainly Ras GTPase binding, ubiquitin-like protein transferase activity, DNA-binding transcription factor binding, ubiquitin-protein transferase activity, nucleoside-triphosphatase regulator activity, transcription coactivator activity, and ubiquitin-like protein ligase binding. Signaling pathways such as MAPK, FoxO, Ras, PIK3-Akt, and Wnt were also enriched.
UNASSIGNED: These findings aid in understanding the mechanisms of diquat poisoning and the related pathways, where DE miRNAs serve as targets for gene therapy.

Sweroside is a natural monoterpene derived from Swertia pseudochinensis Hara. Recently, studies have shown that sweroside exhibits a variety of biological activities, such as anti-inflammatory, antioxidant, and hypoglycemic effects. However, its role and mechanisms in high glucose (HG)-induced renal injury remain unclear. Herein, we established a renal injury model in vitro by inducing human renal tubular epithelial cell (HK-2 cells) injury by HG. Then, the effects of sweroside on HK-2 cell activity, inflammation, reactive oxygen species (ROS) production, and epithelial mesenchymal transition (EMT) were observed. As a result, sweroside treatment ameliorated the viability, inhibited the secretion of inflammatory cytokines (TNF-α, IL-1β, and VCAM-1), reduced the generation of ROS, and inhibited EMT in HK-2 cells. Moreover, the protein expression of SIRT1 was increased and the acetylation of p65 NF-kB was decreased in HK-2 cells with sweroside treatment. More importantly, EX527, an inhibitor of SIRT1, that inactivated SIRT1, abolished the improvement effects of sweroside on HK-2 cells. Our findings suggested that sweroside may mitigate HG-caused injury in HK-2 cells by promoting SIRT1-mediated deacetylation of p65 NF-kB.

Study has demonstrated the abnormal expression and role of lncRNA CASC15 in diabetes patients with chronic renal failure. However, its role in diabetes nephropathy (DN) is still unclear. This study aimed to investigate the potential mechanism and role of lncRNA CASC15 in DN. The relationship between miR-424 and CASC15/SP-A was predicted by Starbase software and verified by luciferase reporter assay. HK-2 cells were treated with 25 mM glucose (HG) for 24 h to establish DN cell model. MTT and flow cytometry analysis were carried out to test cell proliferation and apoptosis. Epithelial-to-mesenchymal transition (EMT) markers were analyzed by RT-qPCR and western blot assay. We proved that CASC15 could interact with miR-424, and SP-A was a target of miR-424. HG-treatment significantly enhanced lncRNA CASC15 level and decreased miR-424 level in HK-2 cells. LncRNA CASC15-siRNA significantly improved cell viability, repressed apoptosis, promoted E-cadherin expression, and inhibited N-cadherin expression in HG-treated HK-2 cells, and these effects were reversed by miR-424 inhibitor. SP-A was highly expressed in HG-treated HK-2 cells. The biological effects of miR-424 mimic on HG-treated HK-2 cells were reversed by SP-A-plasmid. In conclusion, lncRNA CASC15 inhibition relieved HG-induced HK-2 cell injury and EMT through miR-424/SP-A axis.

The biological functions of circTLK1 in acute kidney injury (AKI), which mainly results from renal ischemia-reperfusion (IR), remain largely unknown. HK-2 cell treatment with oxygen and glucose deprivation, reoxygenation, and glucose (OGD/R) was used to simulate an AKI model that was mainly caused by renal IR. Then, the circTLK1 expression level in HK-2 cells treated with OGD/R was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Functional experiments were performed with circTLK1 knockdown of HK-2 cells via Cell Counting Kit-8 (CCK8), flow cytometry (FCM), RT-qPCR, and western blotting. The circTLK1-miRNAs-mRNAs network was constructed following the ceRNA mechanism and visualized by Cytoscape software to investigate the mechanism of circTLK1 in AKI. RT-qPCR was performed to verify the relationship between circTLK1, miR-136-5p, and Bcl2. The level of miR-136-5p was knocked down to ensure its function in OGD/R-triggered apoptosis through experiments, including CCK8, FCM, RT-qPCR, and western blotting. CircTLK1 was downregulated in HK-2 cells subjected to OGD/R treatment and in mouse kidney tissues after renal IR, but the expression of miR-136-5p was the opposite. Interference with circTLK1 expression accelerated HK-2 cell apoptosis, which was overturned by miR-136-5p inhibitors. CircTLK1 targets miR-136-5p to upregulate Bcl2 expression and attenuate apoptosis in HK-2 cells. These data revealed the possible role of circTLK1 as a new biomarker for diagnosis as well as a target in AKI through the miR-136-5p/Bcl2 signaling axis.

BACKGROUND: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multipotent protein that plays essential roles in cellular responses to oxidative stress.
METHODS: To examine the role of APE1/Ref-1 in ischemia-reperfusion (I/R) injuries and hydrogen peroxide (H2O2)-induced renal tubular apoptosis, we studied male C57BL6 mice and human proximal tubular epithelial (HK-2) cells treated with H2O2 at different concentrations. The colocalization of APE1/Ref-1 in the proximal tubule, distal tubule, thick ascending limb, and collecting duct was observed with confocal microscopy. The overexpression of APE1/Ref-1 with knockdown cell lines using an APE1/Ref-1-specific DNA or small interfering RNA (siRNA) was used for the apoptosis assay. The promotor activity of nuclear factor kappa B (NF-κB) was assessed and electrophoretic mobility shift assay was conducted.
RESULTS: APE1/Ref-1 was predominantly localized to the renal tubule nucleus. In renal I/R injuries, the levels of APE1/Ref-1 protein were increased compared with those in kidneys subjected to sham operations. The overexpression of APE1/Ref-1 in HK-2 cells enhanced the Bax/Bcl-2 ratio as a marker of apoptosis. Conversely, the suppression of APE1/Ref-1 expression by siRNA in 1-mM H2O2-treated HK-2 cells decreased the Bax/Bcl-2 ratio, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and NF-κB. In HK-2 cells, the promoter activity of NF-κB increased following H2O2 exposure, and this effect was further enhanced by APE1/Ref-1 transfection.
CONCLUSIONS: The inhibition of APE1/Ref-1 with siRNA attenuated H2O2-induced apoptosis through the modulation of mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 and the nuclear activation of NF-κB and proapoptotic factors.

Diabetes-related vascular complications include diabetic cardiovascular diseases (CVD), diabetic nephropathy (DN) and diabetic retinopathy, etc. DN can promote the process of end-stage renal disease. On the other hand, atherosclerosis accelerates kidney damage. It is really an urge to explore the mechanisms of diabetes-exacerbated atherosclerosis as well as new agents for treatment of diabetes-exacerbated atherosclerosis and the complications. In this study we investigated the therapeutic effects of fisetin, a natural flavonoid from fruits and vegetables, on kidney injury caused by streptozotocin (STZ)-induced diabetic atherosclerosis in low density lipoprotein receptor deficient (LDLR-/-) mice. Diabetes was induced in LDLR-/- mice by injecting STZ, and the mice were fed high-fat diet (HFD) containing fisetin for 12 weeks. We found that fisetin treatment effectively attenuated diabetes-exacerbated atherosclerosis. Furthermore, we showed that fisetin treatment significantly ameliorated atherosclerosis-enhanced diabetic kidney injury, evidenced by regulating uric acid, urea and creatinine levels in urine and serum, and ameliorating morphological damages and fibrosis in the kidney. In addition, we found that the improvement of glomerular function by fisetin was mediated by reducing the production of reactive oxygen species (ROS), advanced glycosylation end products (AGEs) and inflammatory cytokines. Furthermore, fisetin treatment reduced accumulation of extracellular matrix (ECM) in the kidney by inhibiting the expression of vascular endothelial growth factor A (VEGFA), fibronectin and collagens, while enhancing matrix metalloproteinases 2 (MMP2) and MMP9, which was mainly mediated by inactivating transforming growth factor β (TGFβ)/SMAD family member 2/3 (Smad2/3) pathways. In both in vivo and in vitro experiments, we demonstrated that the therapeutic effects of fisetin on kidney fibrosis resulted from inhibiting CD36 expression. In conclusion, our results suggest that fisetin is a promising natural agent for the treatment of renal injury caused by diabetes and atherosclerosis. We reveal that fisetin is an inhibitor of CD36 for reducing the progression of kidney fibrosis, and fisetin-regulated CD36 may be a therapeutic target for the treatment of renal fibrosis.